Ubiquitin-mediated regulation of 3-hydroxy-3-methylglutaryl-CoA reductase

Hampton, Randolph Y.; Bhakta, Hetal

doi:10.1073/pnas.94.24.12944

Cited by 138 publications

(153 citation statements)

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“…We next used the USA1-ΔUBL/hrd3Δ strain to study the role of Hrd3 in ERAD-M independent of effects on Hrd1 stability, starting with the eponymous substrate Hmg2-GFP. Hmg2-GFP undergoes Hrd1-dependent degradation in wild-type cells and is completely stable in either hrd1Δ or hrd3Δ strains (12,13,22). Hmg2-GFP was also completely stabilized by removal of HRD3 in USA1-ΔUBL/hrd3Δ, although Hrd1 levels were unaffected, observed by Chx chase (Figs.…”

Section: Significancementioning

confidence: 95%

Direct and essential function for Hrd3 in ER-associated degradation

Vashistha

Neal

Singh

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The HRD (HMG-CoA reductase degradation) pathway is a conserved route of endoplasmic reticulum-associated degradation (ERAD), by which misfolded ER proteins are ubiquitinated and degraded. ERAD substrates are ubiquitinated by the action of the Hrd1 RING-H2 E3 ligase. Hrd1 is always present in a stoichiometric complex with the ER membrane protein Hrd3, which is also required for HRD-dependent degradation. Despite its conserved presence, unequivocal study of Hrd3 function has been precluded by its central role in Hrd1 stability. Loss of Hrd3 causes unrestricted self-degradation of Hrd1, resulting in significant loss of the core ligase. Accordingly, the degree to which Hrd3 functions independently of Hrd1 stabilization has remained unresolved. By capitalizing on our studies of Usa1 in Hrd1 degradation, we have devised a new approach to evaluate Hrd3 functions in ERAD. We now show that Hrd3 has a direct and critical role in ERAD in addition to Hrd1 stabilization. This direct component of Hrd3 is phenotypically as important as Hrd1 in the native HRD complex. Hrd3 was required the E3 activity of Hrd1, rather than substrate or E2 recruitment to Hrd1. Although Hrd1 can function in some circumstances independent of Hrd3, these studies show an indispensable role for Hrd3 in living cells.highly conserved quality control pathway responsible for the degradation of both misfolded and normal proteins that limits cellular stress and cytotoxicity caused by an accumulation of misfolded ER proteins (1, 2). ERAD occurs through the ubiquitin proteasome pathway, wherein ER-localized ubiquitin ligases covalently modify substrates by attaching multiple copies of the protein ubiquitin, thus tagging them for degradation by cytosolic 26S proteasome (3-5).In Saccharomyces cerevisiae, the highly conserved HRD (HMGCoA † reductase degradation) pathway degrades a variety of ERAD substrates, including misfolded luminal proteins (ERAD-L substrates) such as CPY* and integral membrane proteins (ERAD-M substrates) such as Hmg2 (6, 7). Briefly, ERAD-L substrates are ubiquitinated by the HRD complex, which includes the ubiquitin RING-H2 ligase Hrd1, complex members Hrd3, Usa1, and Der1, and luminal factors implicated in substrate selection (3,(8)(9)(10)(11). Conversely, ERAD-M substrates require only Hrd1 and Hrd3 for in vivo degradation (12, 13). Although Hrd1 functions as the core catalytic subunit of the HRD E3 ligase, Hrd3's function has remained enigmatic. Both Hrd1 and Hrd3 are highly conserved, and Hrd1 is rate limiting for degradation of misfolded substrates (3,12,14,15). In yeast, the steady-state levels of Hrd1 strongly depend on Hrd3. At native levels of the HRD components, Hrd3 is required for Hrd1 stability: In a hrd3Δ, Hrd1 half-life drops to less than 15 min, resulting in very low levels (12). This concomitant loss of Hrd1 is due to rapid autodegradation, catalyzed by the RING domain of Hrd1 (16).Because of Hrd3's strong role in maintaining Hrd1 stability, it has remained unclear whether Hrd3 has any other independent ERAD functions. A ...

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Section: Significancementioning

confidence: 95%

Direct and essential function for Hrd3 in ER-associated degradation

Vashistha

Neal

Singh

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Plasmids and DNA Methods-The following plasmids were previously described: pRH469 (Hmg2p-GFP) (12), pRH244 (6myc-Hmg2p) (13), pRH423 (1myc-Hmg2p) (14), pRH 2038 (TDH3-Der1p) (15), pRH 808 (TDH3-Hrd1p) (6). Plasmids expressing HA-CPY* (pRH1377) and KWW-HA (pRH1960) were obtained from Davis Ng (16).…”

Section: Methodsmentioning

confidence: 99%

Usa1p Is Required for Optimal Function and Regulation of the Hrd1p Endoplasmic Reticulum-associated Degradation Ubiquitin Ligase

Carroll

Hampton

2010

Journal of Biological Chemistry

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Usa1p is a recently discovered member of the HRD ubiquitin ligase complex. The HRD pathway is a conserved route of ubiquitin-dependent, endoplasmic reticulum (ER)-associated degradation (ERAD) of numerous lumenal (ERAD-L) and membrane-anchored (ERAD-M) substrates. We have investigated Usa1p to understand its importance in HRD complex action. Usa1p was required for the optimal function of the Hrd1p E3 ubiquitin ligase; its loss caused deficient degradation of both membrane-associated and lumenal proteins. Furthermore, Usa1p functioned in regulation of Hrd1p by two mechanisms. First, Hrd1p self-degradation, which serves to limit the levels of uncomplexed E3, is absolutely dependent on Usa1p and the ubiquitin-like (Ubl) domain of Usa1p. We found that Usa1p allows Hrd1p degradation by promoting trans interactions between Hrd1p molecules. The Ubl domain of Usa1p was required specifically for Hrd1p self-ubiquitination but not for degradation of either ERAD-L or ERAD-M substrates. In addition, Usa1p was able to attenuate the activity-dependent toxicity of Hrd1p without compromising substrate degradation, indicating a separate role in ligase regulation that operates in parallel to stability control. Many of the described actions of Usa1p are distinct from those of Der1p, which is recruited to the HRD complex by Usa1p. Thus, this novel, conserved factor is broadly involved in the function and regulation of the HRD pathway of ERAD. ER3 -associated degradation (ERAD) is a conserved process by which eukaryotic cells target and degrade ER-resident proteins by the ubiquitin-proteasome pathway. ERAD pathways play a major role in the destruction of misfolded or unassembled ER proteins, including both lumenal and integralmembrane substrates. In addition, normal proteins are regulated by this pathway, the most prominent case being the sterol pathway-regulated degradation of HMG-CoA reductase in both yeast and mammals (1, 2). Eukaryotic ERAD is brought about by the action of multiple pathways of ubiquitin-mediated degradation that operate at the ER surface (3, 4).Covalent addition of ubiquitin to proteins brings about their recognition and degradation by the cytosolic 26S proteasome. Protein ubiquitination occurs by a cascade of enzymes that add 7.6-kDa ubiquitin to the targeted protein. The E1 ubiquitinactivating enzyme first forms a high energy bond with ubiquitin in an ATP-dependent reaction, and then the ubiquitin is transferred to an E2, or ubiquitin-conjugating enzyme. E2-bound ubiquitin is next transferred from the charged E2 to the target protein by the action of a ubiquitin ligase, or E3, that ensures specificity of transfer to the proper degradation substrate. The action of the E3 is iterative, causing the construction of a substrate-bound multiubiquitin chain that is recognized by the 26S proteasome (5). Several E3 ubiquitin ligases are involved in the destruction of ER proteins. Thus, ERAD is a composite of ubiquitination pathways with distinct ligases that use both separate and common components to effect recognition,...

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“…If the proteasome is indeed required for dislocation (8 -11), then ubiquitination of ERAD substrates would not be observed. This applies especially to luminal ERAD substrates, as membrane ERAD substrates inherently display cytosolic domains, which may be ubiquitinated irrespective of dislocation (11)(12)(13)(14)(15). Therefore, it is our view that luminal ERAD substrates, whose dislocation is an absolute prerequisite for ubiquitination, are the proteins of choice to address directly coupling among dislocation, ubiquitination, and proteasomal activity.…”

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confidence: 99%

Distinct Steps in Dislocation of Luminal Endoplasmic Reticulum-associated Degradation Substrates

Elkabetz

Shapira

Rabinovich

et al. 2004

Journal of Biological Chemistry

104

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Dislocation of endoplasmic reticulum-associated degradation (ERAD) substrates from the endoplasmic reticulum (ER) lumen to cytosol is considered to occur in a single step that is tightly coupled to proteasomal degradation. Here we show that dislocation of luminal ERAD substrates occurs in two distinct consecutive steps. The first is passage across ER membrane to the ER cytosolic face, where substrates can accumulate as ubiquitin conjugates. In vivo, this step occurs despite proteasome inhibition but requires p97/Cdc48p because substrates remain entrapped in ER lumen and are prevented from ubiquitination in cdc48 yeast strain. The second dislocation step is the release of accumulated substrates to the cytosol. In vitro, this release requires active proteasome, consumes ATP, and relies on salt-removable ERbound components, among them the ER-bound p97 and ER-bound proteasome, which specifically interact with the cytosol-facing substrates. An additional role for Cdc48p subsequent to ubiquitination is revealed in the cdc48 strain at permissive temperature, consistent with our finding that p97 recognizes luminal ERAD substrates through multiubiquitin. BiP interacts exclusively with ERAD substrates, suggesting a role for this chaperone in ERAD. We propose a model that assigns the cytosolic face of the ER as a midpoint to which luminal ERAD substrates emerge and p97/Cdc48p and the proteasome are recruited. Although p97/Cdc48p plays a dual role in dislocation and is involved both in passage of the substrate across ER membrane and subsequent to its ubiquitination, the proteasome takes part in the release of the substrate from the ER face to the cytosol en route to degradation.The endoplasmic reticulum-associated degradation (ERAD) 1 is a quality control process that selectively eliminates malfolded proteins or unassembled subunits of oligomeric proteins in the secretory pathway (1, 2). ERAD substrates are dislocated from the endoplasmic reticulum (ER) back to the cytosol via the Sec61 complex (3-6). In the cytosol, ubiquitin is conjugated to the ERAD substrates that are degraded by the proteasome (7). The proteolytically active proteasome has been implicated in the dislocation of ERAD substrates by virtue of their scarcity in cytosol of proteasome-inhibited cells (8 -11). Although stabilization of proteins in the secretory pathway by proteasome inhibitors is generally accepted as an indication for ERAD, ubiquitination of such proteins is a direct evidence for the access of the substrate to the cytosol (7). In most cases, however, accumulation of the multiubiquitinated ERAD substrates to detectable levels requires inhibition of the proteasome. If the proteasome is indeed required for dislocation (8 -11), then ubiquitination of ERAD substrates would not be observed. This applies especially to luminal ERAD substrates, as membrane ERAD substrates inherently display cytosolic domains, which may be ubiquitinated irrespective of dislocation (11-15). Therefore, it is our view that luminal ERAD substrates, whose dislocation is an ...

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Ubiquitin-mediated regulation of 3-hydroxy-3-methylglutaryl-CoA reductase

Cited by 138 publications

References 29 publications

Direct and essential function for Hrd3 in ER-associated degradation

Direct and essential function for Hrd3 in ER-associated degradation

Usa1p Is Required for Optimal Function and Regulation of the Hrd1p Endoplasmic Reticulum-associated Degradation Ubiquitin Ligase

Distinct Steps in Dislocation of Luminal Endoplasmic Reticulum-associated Degradation Substrates

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